Na+/H+ exchangers control the movement of salt, water and acid-base equivalents, and play a critical role in pH regulation, cell proliferation, volume control and ion homeostasis. A wide range of clinical conditions are associated with derangements in Na+/H+ exchange, including hypertension, cardiac ischemia and various acid base disorders. Although a number of plasma membrane NHE isoforms have been described in higher vertebrates, endosomal Na+/H+ exchangers represent a physiologically important sub-type that have only recently been identified at a molecular level. We have cloned, expressed, and localized novel endosomal exchangers from yeast, Nhx1, and from human, NHE6, as a starting point to explore the cellular and physiological role of intracellular Na+/H+ exchange. We will begin by determining the transport characteristics of human NHE6, including ion selectivity, pH sensitivity and pharmacological profile, using 22Na transport and pH-sensitive fluorescent indicators targeted to the endosomal lumen (Aim 1). These studies will establish whether NHE6 serves as a H+ leak pathway, to limit vesicular acidification and sequester osmotically active ions, as has been reported for endosomal exchangers from yeast and plants. The molecular determinants of ion binding and transport in exchangers remain largely unknown. We will examine structure-function relations in Nhx1, as a model of the NHE family, using phenotype screening in the genetically amenable model organism, yeast (Aim 2). A combination of directed and localized random mutagenesis will be used to target membrane helices believed to be important for transport. Loss-of-function in plasmid-encoded mutants will be identified by assessing salt or hygromycin sensitivity in yeast strains engineered to lack chromosomal Nhx 1. Second site mutations that confer regain-of-function are likely to reveal inter- and intra-domain interactions between side chains. These studies will help define the ion transport pathway. The C-terminal domains of Nhx1 and NHE6 are smaller and more divergent than those of other NHE, suggesting that they may interact with distinct regulatory proteins for endosome-specific functions. We have identified several candidate proteins by yeast 2-hybrid approaches and will pursue detailed studies or them to determine a functional role (Aim 3). In summary, we propose to elucidate cellular function and mode of regulation of two novel members of a physiologically important, clinically relevant and mechanistically interesting family of transport proteins by drawing on the combined and complementary strengths of yeast and mammalian cells in culture.